Torin2 overcomes sorafenib resistance via suppressing mTORC2-AKT-BAD pathway in hepatocellular carcinoma cells

Background:Sorafenib is an oral multi-kinase inhibitor that was approved by the US Food and Drug Administration for the treatment of patients with advanced hepatocellular carcinoma(HCC).However,resistance to sorafenib is an urgent problem to be resolved to improve the therapeutic efficacy of sorafenib.As the activation of AKT/mTOR played a pivotal role in sorafenib resistance,we evaluated the effect of a dual mTOR complex 1/2 inhibitor Torin2 on overcoming the sorafenib resistance in HCC cells.Methods:The sorafenib-resistant Huh7 and Hep3B cell lines were established from their parental cell lines.The synergistic effect of sorafenib and Torin2 on these cells was measured by cell viability assay and quantified using the Chou-Talalay method.Apoptosis induced by the combination of sorafenib and Torin2 and the alteration in the specific signaling pathways of interest were detected by Western blotting.Results:Sorafenib treatment inversely inhibited AKT in parental but activated AKT in sorafenib-resistant Huh7 and Hep3B HCC cells,which underscores the significance of AKT activation.Torin2 and sorafenib synergistically suppressed the viability of sorafenib-resistant cells via apoptosis induction.Torin2 successfully suppressed the sorafenib-activated mTORC2-AKT axis,leading to the dephosphorylation of Ser136 in BAD protein,and increased the expression of total BAD,which contributed to the apoptosis in sorafenibresistant HCC cells.Conclusions:In this study,Torin2 and sorafenib showed synergistic cytostatic capacity in sorafenibresistant HCC cells,via the suppression of mTORC2-AKT-BAD pathway.Our results suggest a novel strategy of drug combination for overcoming sorafenib resistance in HCC.

Silencing MFN2 Drives WNT/β-catenin Nucleation to Reduce Sorafenib Sensitivity in Hepatocellular Carcinoma Cells

Objective Mitofusin-2(MFN2)is a mitochondrial membrane protein that plays a critical role in regulating mitochondrial fusion and cellular metabolism.To further elucidate the impact of MFN2,this study aimed to investigate its significance on hepatocellular carcinoma(HCC)cell function and its potential role in mediating chemosensitivity.Methods This study investigated the effects of silencing and overexpressing MFN2 on the survival,proliferation,invasion and migration abilities,and sorafenib resistance of MHCC97-L HCC cells.Additional experiments were conducted using XAV939(aβ-catenin inhibitor)and HLY78(aβ-catenin activator)to further validate these findings.Results Silencing MFN2 significantly promoted the survival and proliferation of MHCC97-L cells,enhanced their invasion and migration capacities,increased the IC50 of sorafenib,reduced the percentage of TUNEL-positive cells,and decreased the expression of proapoptotic proteins.Additionally,silencing MFN2 markedly induced the nuclear translocation ofβ-catenin,increasedβ-catenin acetylation levels and enhanced the expression of the downstream regulatory proteins Snail1 and Vimentin while inhibiting E-cadherin expression.Conversely,overexpressing MFN2 reversed the effects observed in MHCC97-L cells mentioned above.The results confirmed that silencing MFN2 activated theβ-catenin/epithelial-mesenchymal transition(EMT)pathway and reduced the sensitivity of cells to sorafenib,which could be reversed by XAV939 treatment.Conversely,overexpression of MFN2 inhibited theβ-catenin/EMT pathway and increased the sensitivity of cells to sorafenib,which could be altered by HLY78.Conclusion Low expression of MFN2 in HCC cells promotes the nuclear translocation ofβ-catenin,thereby activating the EMT pathway and mediating resistance to sorafenib.

Microvesicles:the functional mediators in sorafenib resistance

Overcoming drug resistance in cancer therapies remains challenging,and the tumor microenvironment plays an important part in it.Microvesicles(MVs)are functional natural carriers of cellular information,participate in intercellular communication,and dynamically regulate the tumor microenvironment.They contribute to drug resistance by transferring functional molecules between cells.Conversely,due to their specific cell or tissue targeting ability,MVs are considered as carriers for therapeutic molecules to reverse drug resistance.Thus,in this mini-review,we aim to highlight the crucial role of MVs in cell-to-cell communication and therefore their diverse impact mainly on liver cancer progression and treatment.In addition,we summarize the possible mechanisms for sorafenib resistance(one of the main hurdles in hepatocellular carcinoma treatments)and recent advances in using MVs to reverse sorafenib resistance in liver cancer therapies.Identifying the functional role of MVs in cancer therapy might provide a new aspect for developing precise novel therapeutics in the future.

Physcion increases the sensitivity of hepatocellular carcinoma to sorafenib through miRNA-370/PIM1 axis-regulated glycolysis

BACKGROUND Resistance to sorafenib has become a challenge in clinical treatment of hepatocellular carcinoma(HCC).Physcion is a common bioactive anthraquinone that has potential as an anticancer agent.AIM To study the effect of physcion on sensitizing HCC cells to sorafenib.METHODS Sorafenib-resistant HCC cells were established and treated with sorafenib and/or physcion.The cell viability,proliferation and apoptosis were measured by cell counting kit-8,colony formation,flow cytometry,and in vivo xenograft model.Glucose uptake,lactate acid production,extracellular acidification rate(ECAR),and oxygen consumption rate(OCR)were measured to analyze glycolysis.Expression of glycolysis-related regulators was assessed by western blotting.RESULTS The addition of physcion significantly enhanced the antitumor effects of sorafenib on sorafenib-resistant HCC cells,manifested by enhanced apoptosis and suppressed cell growth.The glucose uptake,lactate acid production,and ECAR were elevated,and OCR was suppressed by physcion treatment.The level of PIM1 was elevated and miR-370 was suppressed in sorafenib-resistant HCC cells compared with the parental cells,which was suppressed by physcion treatment.Inhibition of miR-370 notably reversed the effects of physcion on sorafenib-resistant HCC cells.CONCLUSION Our data indicated that physcion enhanced the sensitivity of HCC cells to sorafenib by enhancing miR-370 to suppress PIM1-promoted glycolysis.

LINC00662 affects the sensitivity of hepatocellular carcinoma cells to sorafenib drug by regulating miR-106a-5p/CAV1 axis

Objective:To investigate the mechanism of long non-coding RNA-LINC00662 on induction of sorafenib resistance in hepatocellular carcinoma(HCC)cells.Methods:HCC cells(HepG2,HCCLM3),sorafenib-resistant hepatocellular carcinoma cells HCC-SR(HepG2-SR,HCCLM3-SR)were investigated by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and Western blot was used to detect LINC00662,miR-106a-5p and cavitin-1(CAV1)expression in each group of cells.106a-5p and cavitin-1(CAV1)expression levels were measured by RT-qPCR and Western blot.The si-LINC00662 and miR-106a-5p mimics were transfected with HCC-SR cells,respectively,and cell sensitivity to sorafenib drug was detected by cell activity kit(CCK-8).And the targeting relationship between LINC00662 and miR-106a-5p,miR-106a-5p and CAV1 was further determined by dual luciferase reporter assay,RT-qPCR,and Western blot.Results:The relative expression of LINC00662 and CAV1 was significantly increased and miR-106a-5p expression was significantly decreased in HCC-SR cells(P<0.01,P<0.001);interference with LINC00662 expression or overexpression of miR-106a-5p significantly increased the sensitivity of HCC-SR cells to sorafenib drug(P<0.05,P<0.01).And LINC00662 targeted to negatively regulate miR-106a-5p expression and miR-106a-5p targeted to negatively regulate CAV1 expression(P<0.05).Conclusion:LINC00662 could act as a competitive endogenous RNA(ceRNA)of miR-106a-5p to promote the expression of CAV1 and mediate the resistance of sorafenib in HCC cells.Interfering with LINC00662 expression can inhibit sorafenib resistance and increase sorafenib drug sensitivity in HCC cells.

Identification of novel immune ferroptosis-specific genes associated with prognostic features in hepatocellular carcinoma

Background:As the most common iron metabolism and storage organ,the specific regulatory mechanism and prognostic relevance of hepatocellular carcinoma(HCc)need further study.Objective:We aimed to perform a multi-omics integration and a ferroptosis-associated signature of HCC.Methods:Gene Expression Omnibus was searched for available data with ferroptosis inducers,and three independent datasets(GSE104462,GSE112384,and GSE142591)were collected.Multi-omic data with available clinical information of TCGA-LIHC and CHCC were retrieved from the Cancer Genome Atlas(TCGA,http://cancergenome.nih.gov/)and iProX database(www.iprox.org,IPx0000937000).Integrated multi-omics analyses revealed the difference among biological functions,the activation status of key signaling pathways,tumor microenvironment,and sorafenib resistance in HCC.Single-sample gene set enrichment analysis(ssGSEA)identified a novel panel associated with clinical and molecular attributes,including survival,immune microenvironment,sorafenib resistance,genetic profile,liver-specific proteome,and phosphoproteome.Results:We performed a multi-omics integrationfor ferroptosis-associated characterization of HCC using paired tumor and adjacent liver tissues from 370 samples of TCGA.We proposed a novel panel including twelve genes that could reflect the prognosis and capacity of ferroptosis(ATAD2,DTL,DUT,E2F2,GINS2,FOXK1,MCM4,MCM5,MTHFD1,PHF19,POLA1,THOC6).Samples with high ferroptosis prognostic scores suggested significantly inferior survival(P=0.0028),a lower degree of ferroptosis(P<0.001),and greater ferroptosis induction capacity(P<0.001).Conclusion:The score in our study revealed the inherent state and further potential of ferroptosis in tumor cells,which also distinct features in tumor metabolism,microenvironment,clinical phenotype,and potential therapeutics.

Radiomics signature:A potential biomarker forβ-arrestin1 phosphorylation prediction in hepatocellular carcinoma

BACKGROUND The phosphorylation status ofβ-arrestin1 influences its function as a signal strongly related to sorafenib resistance.This retrospective study aimed to develop and validate radiomics-based models for predictingβ-arrestin1 phosphorylation in hepatocellular carcinoma(HCC)using whole-lesion radiomics and visual imaging features on preoperative contrast-enhanced computed tomography(CT)images.AIM To develop and validate radiomics-based models for predictingβ-arrestin1 phosphorylation in HCC using radiomics with contrast-enhanced CT.METHODS Ninety-nine HCC patients(training cohort:n=69;validation cohort:n=30)receiving systemic sorafenib treatment after surgery were enrolled in this retrospective study.Three-dimensional whole-lesion regions of interest were manually delineated along the tumor margins on portal venous CT images.Radiomics features were generated and selected to build a radiomics score using logistic regression analysis.Imaging features were evaluated by two radiologists independently.All these features were combined to establish clinico-radiological(CR)and clinico-radiological-radiomics(CRR)models by using multivariable logistic regression analysis.The diagnostic performance and clinical usefulness of the models were measured by receiver operating characteristic and decision curves,and the area under the curve(AUC)was determined.Their association with prognosis was evaluated using the Kaplan-Meier method.RESULTS Four radiomics features were selected to construct the radiomics score.In the multivariate analysis,alanine aminotransferase level,tumor size and tumor margin on portal venous phase images were found to be significant independent factors for predictingβ-arrestin1 phosphorylation-positive HCC and were included in the CR model.The CRR model integrating the radiomics score with clinico-radiological risk factors showed better discriminative performance(AUC=0.898,95%CI,0.820 to 0.977)than the CR model(AUC=0.794,95%CI,0.686 to 0.901;P=0.011),with increased clinical usefulness confirmed in both the training and validation cohorts using decision curve analysis.The risk ofβ-arrestin1 phosphorylation predicted by the CRR model was significantly associated with overall survival in the training and validation cohorts(log-rank test,P<0.05).CONCLUSION The radiomics signature is a reliable tool for evaluatingβ-arrestin1 phosphorylation which has prognostic significance for HCC patients,providing the potential to better identify patients who would benefit from sorafenib treatment.

CXCR4-guided liposomes regulating hypoxic and immunosuppressive microenvironment for sorafenib-resistant tumor treatment

Clinical sorafenib treatment could activate C-X-C receptor type 4(CXCR4)/stromal source factor-1α(SDF-1α)axis to aggravate intra-tumoral hypoxia of hepatocellular carcinoma(HCC),which further leads to progression,invasion,metastasis,and immunosuppression of tumors and in return causes resistance to sorafenib therapy.Therefore,a multi-functional oxygen delivery nanoplatform was rationally constructed based on an oxygen-saturated perfluorohexane(PFH)-cored liposome,with the CXCR4 antagonist LFC131 peptides modifying on the surface to simultaneously deliver sorafenib and the CSF1/CSF1R inhibitor PLX3397(named PFH@LSLP)for sorafenib-resistant HCC treatment.The PFH@LSLP was developed to overcome sorafenib resistance by syner-gistic effects of the following 3 roles:1)the O_(2)-saturated PFH core could alleviate the tumor hypoxia by O_(2) supply;2)the LFC131 peptide recognized the hypoxia-related overexpressed CXCR4 and then blocked SDF-1α/CXCR4 axis to re-sensitize the HCC cells to sorafenib;3)PLX3397 activated the immune responses via inhibiting the CSF1/CSF1R pathway in TAMs,further enhanced CD8^(+)T cell infiltration to reverse immunosuppression in tumors.Antitumor performance on H22 tumor-bearing mice and HCC patient-derived tumor xenograft(PDX)model showed that PFH@LSLP could overcome sorafenib resistance by synergistic effect of hypoxia attenuation,resistance-related gene regulation,and immune-microenvironment modification.

Overexpression of RBM34 Promotes Tumor Progression and Correlates with Poor Prognosis of Hepatocellular Carcinoma

Background and Aims:Emerging evidence suggests that RNA-binding motif(RBM)proteins are involved in hepatocarcinogenesis and act either as oncogenes or tumor suppressors.The objective of this study was to investigate the role of RBM34,an RBM protein,in hepatocellular carcinoma(HCC).Methods:We first examined the expression of RBM34 across cancers.The correlation of RBM34 with clinicopathological features and the prognostic value of RBM34 for HCC was then investigated.Functional enrichment analysis of RBM34-related differentially expressed genes(DEGs)was performed to explore its biological function.RNA sequencing(RNA-seq)was applied to identify downstream genes and pathways affected upon RBM34 knockout.The correlation of RBM34 with immune characteristics was also analyzed.The oncogenic function of RBM34 was examined in in vitro and in vivo experiments.Results:RBM34 was highly expressed in hepatocellular carcinoma and correlated with poor clinicopathological features and prognosis.RBM34 was positively associated with tumor immune cell infiltration,biomarkers of immune cells,and immune checkpoint expression.A positive correlation was also observed between RBM34,T cell exhaustion,and regulatory T cell marker genes.Knockout of RBM34 significantly inhibited cell proliferation,migration,and xenograft tumor growth,and sensitized HCC cells to sorafenib treatment.RBM34 inhibition reduced FGFR2 expression and affected PI3K-AKT pathway activation in HCC cells.Conclusions:Our study suggests that RBM34 may serve as a new prognostic marker and therapeutic target of HCC.

A biomimetic liver cancer on-a-chip reveals a critical role of LIPOCALIN-2 in promoting hepatocellular carcinoma progression

Hepatic stellate cells(HSCs)represent a significant component of hepatocellular carcinoma(HCC)microenvironments which play a critical role in tumor progression and drug resistance.Tumor-ona-chip technology has provided a powerful in vitro platform to investigate the crosstalk between activated HSCs and HCC cells by mimicking physiological architecture with precise spatiotemporal control.Here we developed a tri-cell culture microfluidic chip to evaluate the impact of HSCs on HCC progression.Onchip analysis revealed activated HSCs contributed to endothelial invasion,HCC drug resistance and natural killer(NK)cell exhaustion.Cytokine array and RNA sequencing analysis were combined to indicate the iron-binding protein LIPOCALIN-2(LCN-2)as a key factor in remodeling tumor microenvironments in the HCC-on-a-chip.LCN-2 targeted therapy demonstrated robust anti-tumor effects both in vitro 3D biomimetic chip and in vivo mouse model,including angiogenesis inhibition,sorafenib sensitivity promotion and NK-cell cytotoxicity enhancement.Taken together,the microfluidic platform exhibited obvious advantages in mimicking functional characteristics of tumor microenvironments and developing targeted therapies.